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US5605662A - Active programmable electronic devices for molecular biological analysis and diagnostics - Google Patents

Active programmable electronic devices for molecular biological analysis and diagnostics Download PDF

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Publication number
US5605662A
US5605662A US08/146,504 US14650493A US5605662A US 5605662 A US5605662 A US 5605662A US 14650493 A US14650493 A US 14650493A US 5605662 A US5605662 A US 5605662A
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US
United States
Prior art keywords
electronic device
electrode
micro
permeation layer
locations
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US08/146,504
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English (en)
Inventor
Michael J. Heller
Eugene Tu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gamida for Life BV
Nanogen Inc
Original Assignee
Nanogen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanogen Inc filed Critical Nanogen Inc
Priority to US08/146,504 priority Critical patent/US5605662A/en
Assigned to NANOGEN, INC. reassignment NANOGEN, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HELLER, MICHAEL J., TU, EUGENE
Priority to US08/271,882 priority patent/US6017696A/en
Priority to US08/304,657 priority patent/US5632957A/en
Priority to CA002175483A priority patent/CA2175483C/en
Priority to CNA2006101003286A priority patent/CN1920055A/zh
Priority to CA002477138A priority patent/CA2477138C/en
Priority to EP01106838A priority patent/EP1120155A3/en
Priority to AT95900430T priority patent/ATE211259T1/de
Priority to NZ500373A priority patent/NZ500373A/en
Priority to PCT/US1994/012270 priority patent/WO1995012808A1/en
Priority to ES95900430T priority patent/ES2170133T3/es
Priority to EP01106841A priority patent/EP1120469A3/en
Priority to PT95900430T priority patent/PT727045E/pt
Priority to JP7513281A priority patent/JPH09504910A/ja
Priority to BR9407952A priority patent/BR9407952A/pt
Priority to CA002504343A priority patent/CA2504343A1/en
Priority to NZ330036A priority patent/NZ330036A/xx
Priority to NZ330037A priority patent/NZ330037A/xx
Priority to DE69429535T priority patent/DE69429535T2/de
Priority to AU81257/94A priority patent/AU692800B2/en
Priority to CNA2004100040679A priority patent/CN1558207A/zh
Priority to CNB941947599A priority patent/CN1142821C/zh
Priority to EP01106840A priority patent/EP1120156A3/en
Priority to EP95900430A priority patent/EP0727045B1/en
Priority to EP01106846A priority patent/EP1120157A3/en
Priority to NZ275962A priority patent/NZ275962A/en
Priority to DK95900430T priority patent/DK0727045T3/da
Priority to US08/534,454 priority patent/US5849486A/en
Priority to FI961843A priority patent/FI961843A/sv
Priority to US08/725,976 priority patent/US5929208A/en
Priority to US08/726,278 priority patent/US6238624B1/en
Priority to US08/753,962 priority patent/US6287517B1/en
Priority to US08/760,933 priority patent/US6652808B1/en
Application granted granted Critical
Publication of US5605662A publication Critical patent/US5605662A/en
Priority to US08/846,876 priority patent/US6309601B1/en
Priority to US08/855,058 priority patent/US6048690A/en
Priority to US08/859,644 priority patent/US6582660B1/en
Priority to US08/986,065 priority patent/US6051380A/en
Priority to US09/141,286 priority patent/US6245508B1/en
Priority to HK98111839A priority patent/HK1011079A1/xx
Priority to US09/203,935 priority patent/US6319472B1/en
Priority to US09/204,324 priority patent/US6638482B1/en
Priority to US09/203,725 priority patent/US6309602B1/en
Priority to US09/203,730 priority patent/US6375899B1/en
Priority to US09/240,931 priority patent/US6315953B1/en
Priority to US09/240,920 priority patent/US6254827B1/en
Priority to US09/240,489 priority patent/US6225059B1/en
Priority to US09/239,598 priority patent/US6331274B1/en
Priority to US09/239,569 priority patent/US6068818A/en
Priority to US09/291,129 priority patent/US6468742B2/en
Priority to US09/354,931 priority patent/US6306348B1/en
Priority to US09/358,788 priority patent/US8389212B1/en
Priority to US09/436,311 priority patent/US6569382B1/en
Priority to US09/444,539 priority patent/US6518022B1/en
Priority to US09/490,965 priority patent/US7172864B1/en
Priority to US09/527,069 priority patent/US6423271B1/en
Priority to US09/532,364 priority patent/US6540961B1/en
Priority to US09/596,657 priority patent/US6726880B1/en
Priority to US09/597,866 priority patent/US7101661B1/en
Priority to US09/849,119 priority patent/US7425308B2/en
Priority to US09/849,122 priority patent/US7241419B2/en
Priority to US09/912,014 priority patent/US20030059929A1/en
Priority to US09/927,820 priority patent/US20010052976A1/en
Priority to US09/952,459 priority patent/US6821729B2/en
Priority to US10/029,472 priority patent/US20020085954A1/en
Priority to US10/170,172 priority patent/US20030190632A1/en
Priority to US10/245,206 priority patent/US7582421B2/en
Priority to US10/371,066 priority patent/US20030162214A1/en
Priority to US10/686,440 priority patent/US20040077074A1/en
Priority to US11/401,713 priority patent/US8630807B2/en
Priority to US11/505,679 priority patent/US7704726B2/en
Priority to JP2006273187A priority patent/JP2007057540A/ja
Priority to US11/726,520 priority patent/US8114589B2/en
Priority to US11/775,724 priority patent/US7858034B2/en
Priority to US11/963,317 priority patent/US8313940B2/en
Assigned to PORTSIDE GROWTH & OPPORTUNITY FUND, AS COLLATERAL AGENT reassignment PORTSIDE GROWTH & OPPORTUNITY FUND, AS COLLATERAL AGENT GRANT OF SECURITY INTEREST Assignors: NANOGEN, INC.
Assigned to ELITECH HOLDING B.V. reassignment ELITECH HOLDING B.V. ORDER RELEASING SECURITY AGREEMENTS AND AUTHORIZING ASSIGNMENT Assignors: PORTSIDE GROWTH & OPPORTUNITY FUND
Assigned to GAMIDA FOR LIFE, B.V. reassignment GAMIDA FOR LIFE, B.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ELITECH HOLDING B.V.
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
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    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • This invention pertains to the design, fabrication, and uses of a self-addressable, self-assembling microelectronic system which can actively carry out and control multi-step and multiplex reactions in microscopic formats.
  • these reactions include molecular biological reactions, such as nucleic acid hybridizations, antibody/antigen reactions, clinical diagnostics, and biopolymer synthesis.
  • Molecular biology comprises a wide variety of techniques for the analysis of nucleic acid and protein, many of which form the basis of clinical diagnostic assays. These techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and separation and purification of nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).
  • Nucleic acid hybridization analysis generally involves the detection of a very small numbers of specific target nucleic acids (DNA or RNA) with probes among a large amount of non-target nucleic acids.
  • hybridization is normally carried out under the most stringent condition, achieved through a combination of temperature, salts, detergents, solvents, chaotropic agents, and denaturants.
  • nucleic acid hybridization formats and stringency control methods it remains difficult to detect low copy number (i.e., 1-100,000) nucleic acid targets even with the most sensitive reporter groups (enzyme, fluorophores, radioisotopes, etc.) and associated detection systems (fluorometers, luminometers, photon counters, scintillation counters, etc.).
  • Hybridization reactions are usually carried out under the most stringent conditions in order to achieve the highest degree of specificity.
  • Methods of stringency control involve primarily the optimization of temperature, ionic strength, and denaturants in hybridization and subsequent washing procedures.
  • the application of these stringency conditions causes a significant decrease in the number of hybridized probe/target complexes for detection.
  • the second problem relates to the high complexity of DNA in most samples, particularly in human genomic DNA samples.
  • a sample is composed of an enormous number of sequences which are closely related to the specific target sequence, even the most unique probe sequence has a large number of partial hybridizations with non-target sequences.
  • the third problem relates to the unfavorable hybridization dynamics between a probe and its specific target. Even under the best conditions, most hybridization reactions are conducted with relatively low concentrations of probes and target molecules. In addition, a probe often has to compete with the complementary strand for the target nucleic acid.
  • the fourth problem for most present hybridization formats is the high level of non-specific background signal. This is caused by the affinity of DNA probes to almost any material.
  • PCR polymerase chain reaction
  • a distinctive exception to the general difficulty in detecting low copy number target nucleic acid with a direct probe is the in-situ hybridization technique.
  • This technique allows low copy number unique nucleic acid sequences to be detected in individual cells.
  • target nucleic acid is naturally confined to the area of a cell ( ⁇ 20-50 ⁇ m 2 ) or a nucleus ( ⁇ 10 ⁇ m 2 ) at a relatively high local concentration.
  • the probe/target hybridization signal is confined to a morphologically distinct area; this makes it easier to distinguish a positive signal from artificial or non-specific signals than hybridization on a solid support.
  • Mimicking the in-situ hybridization new techniques are being developed for carrying out multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip.
  • These hybridization formats are micro-scale versions of the conventional "dot blot" and "sandwich” hybridization systems.
  • the micro-formatted hybridization can be used to carry out "sequencing by hybridization” (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992).
  • SBH makes use of all possible n-nucleotide oligomers (n-mers) to identify n-mers in an unknown DNA sample, which are subsequently aligned by algorithm analysis to produce the DNA sequence (R. Drmanac and R. Crkvenjakov, Yugoslav Patent Application #570/87, 1987; R. Drmanac et al., 4 Genomics, 114, 1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1991; and R. Drmanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13, 1993).
  • the first format involves creating an array of all possible n-mers on a support, which is then hybridized with the target sequence.
  • the second format involves attaching the target sequence to a support, which is sequentially probed with all possible n-mers. Both formats have the fundamental problems of direct probe hybridizations and additional difficulties related to multiplex hybridizations.
  • Southern United Kingdom Patent Application GB 8810400, 1988; E. M. Southern et al., 13 Genomics 1008, 1992, proposed using the first format to analyze or sequence DNA.
  • Southern identified a known single point mutation using PCR amplified genomic DNA.
  • Southern also described a method for synthesizing an array of oligonucleotides on a solid support for SBH.
  • Southern did not address how to achieve optimal stringency condition for each oligonucleotide on an array.
  • Fodor et al. 364 Nature, pp. 555-556, 1993, used an array of 1,024 8-mer oligonucleotides on a solid support to sequence DNA.
  • the target DNA was a fluorescently labeled single-stranded 12-mer oligonucleotide containing only nucleotides A and C. 1 pmol ( ⁇ 6 ⁇ 10 11 molecules) of the 12-mer target sequence was necessary for the hybridization with the 8-mer oligomers on the array. The results showed many mismatches.
  • Fodor et al. did not address the underlying problems of direct probe hybridization, such as stringency control for multiplex hybridizations. These problems, together with the requirement of a large quantity of the simple 12-mer target, indicate severe limitations to this SBH format.
  • Drmanac et al. 260 Science 1649-1652, 1993, used the second format to sequence several short (116 bp) DNA sequences.
  • Target DNAs were attached to membrane supports ("dot blot" format).
  • Each filter was sequentially hybridized with 272 labeled 10-mer and 11-mer oligonucleotides.
  • a wide range of stringency condition was used to achieve specific hybridization for each n-mer probe; washing times varied from 5 minutes to overnight, and temperatures from 0° C. to 16° C. Most probes required 3 hours of washing at 16° C.
  • the filters had to be exposed for 2 to 18 hours in order to detect hybridization signals.
  • the overall false positive hybridization rate was 5% in spite of the simple target sequences, the reduced set of oligomer probes, and the use of the most stringent conditions available.
  • Nucleic acids of different size, charge, or conformation are routinely separated by electrophoresis techniques which can distinguish hybridization species by their differential mobility in an electric field.
  • Pulse field electrophoresis uses an arrangement of multiple electrodes around a medium (e.g., a gel) to separate very large DNA fragments which cannot be resolved by conventional gel electrophoresis systems (see R. Anand and E. M. Southern in Gel Electrophoresis of Nucleic Acids--A Practical Approach, 2 ed., D. Rickwood and B. D. Hames Eds., IRL Press, New York, pp. 101-122, 1990).
  • Pace U.S. Pat. No. 4,908,112, Mar. 13, 1990, teaches using micro-fabrication techniques to produce a capillary gel electrophoresis system on a silicon substrate. Multiple electrodes are incorporated into the system to move molecules through the separation medium within the device.
  • AC alternating current
  • DC direct current
  • the dielectrophoresis process requires a very high frequency AC (1 MHz) voltage and a low conductivity medium. While these techniques can orient DNA molecules of different sizes along the AC field lines, they cannot distinguish between hybridization complexes of the same size.
  • the present invention relates to the design, fabrication, and uses of a self-addressable self-assembling microelectronic system and device which can actively carry out controlled multi-step and multiplex reactions in microscopic formats. These reactions include, but are not limited to, most molecular biological procedures, such as nucleic acid hybridization, antibody/antigen reaction, and related clinical diagnostics.
  • the device is able to carry out multi-step combinational biopolymer synthesis, including, but not limited to, the synthesis of different oligonucleotides or peptides at specific micro-locations.
  • the device is fabricated using both microlithographic and micro-machining techniques.
  • the device has a matrix of addressable microscopic locations on its surface; each individual micro-location is able to electronically control and direct the transport and attachment of specific binding entities (e.g., nucleic acids, antibodies) to itself. All micro-locations can be addressed with their specific binding entities.
  • specific binding entities e.g., nucleic acids, antibodies
  • the device is able to control and actively carry out a variety of assays and reactions.
  • Analytes or reactants can be transported by free field electrophoresis to any specific micro-location where the analytes or reactants are effectively concentrated and reacted with the specific binding entity at said micro-location.
  • the sensitivity for detecting a specific analyte or reactant is improved because of the concentrating effect. Any un-bound analytes or reactants can be removed by reversing the polarity of a micro-location.
  • the device also improves the specificity of assays and reactions.
  • the device provides independent stringency control for hybridization reactions at specific micro-locations.
  • all the micro-locations on the matrix can have different stringency conditions at the same time, allowing multiple hybridizations to be conducted at optimal conditions.
  • the device also facilitates the detection of hybridized complexes at each micro-location by using an associated optical (fluorescent or spectrophotometric) imaging detector system or an integrated sensing component.
  • the active nature of the device allows complex multi-step reactions to be carried out with minimal outside physical manipulations. If desired, a master device addressed with specific binding entities can be electronically replicated or copied to another base device.
  • the disclosed device can carry out multi-step and multiplex reactions with complete and precise electronic control, preferably with a micro-processor.
  • the rate, specificity, and sensitivity of multi-step and multiplex reactions are greatly improved at specific micro-locations of the disclosed device.
  • the present invention overcomes the limitations of the arrays and devices for multi-sample hybridizations described in the background of the invention. Previous methods and devices are functionally passive regarding the actual hybridization process. While sophisticated photolithographic techniques were used to make an array, or microelectronic sensing elements were incorporated for detection, previous devices did not control or influence the actual hybridization process. They are not designed to actively overcome any of the underlying physical problems associated with hybridization reactions.
  • This invention may utilize micro-locations of any size or shape consistent with the objective of the invention.
  • micro-locations in the sub-millimeter range are used.
  • a specific binding entity is generally meant a biological or synthetic molecule that has specific affinity to another molecule, through covalent bonding or non-covalent bonding.
  • a specific binding entity contains (either by nature or by modification) a functional chemical group (primary amine, sulfhydryl, aldehyde, etc.), a common sequence (nucleic acids), an epitope (antibodies), a hapten, or a ligand, that allows it to covalently react or non-covalently bind to a common functional group on the surface of a micro-location.
  • Specific binding entities include, but are not limited to: deoxyribonucleic acids (DNA), ribonucleic acids (RNA), synthetic oligonucleotides, antibodies, proteins, peptides, lectins, modified polysaccharides, synthetic composite macromolecules, functionalized nanostructures, synthetic polymers, modified/blocked nucleotides/nucleosides, modified/blocked amino acids, fluorophores, chromophores, ligands, chelates and haptens.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • synthetic oligonucleotides antibodies
  • proteins proteins
  • peptides lectins
  • modified polysaccharides synthetic composite macromolecules
  • functionalized nanostructures synthetic polymers
  • modified/blocked nucleotides/nucleosides modified/blocked amino acids
  • fluorophores chromophores
  • ligands ligands
  • chelates and haptens
  • stringency control is meant the ability to discriminate specific and non-specific binding interactions.
  • the present invention features a device with an array of electronically self-addressable microscopic locations.
  • Each microscopic location contains an underlying working direct current (DC) micro-electrode supported by a substrate.
  • the surface of each micro-location has a permeation layer for the free transport of small counter-ions, and an attachment layer for the covalent coupling of specific binding entities.
  • array or “matrix” is meant an arrangement of locations on the device.
  • the locations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats.
  • the number of locations can range from several to at least hundreds of thousands.
  • this invention features a method for transporting the binding entity to any specific microlocation on the device.
  • a micro-location When activated, a micro-location can affect the free field electrophoretic transport of any charged functionalized specific binding entity directly to itself.
  • the functionalized specific binding entity Upon contacting the specific micro-location, the functionalized specific binding entity immediately becomes covalently attached to the attachment layer surface of that specific micro-location.
  • Other micro-locations can be simultaneously protected by maintaining them at the opposite potential to the charged molecules. The process can be rapidly repeated until all the micro-locations are addressed with their specific binding entities.
  • charged functionalized specific binding entity is meant a specific binding entity that is chemically reactive (i.e., capable of covalent attachment to a location) and carrying a net change (either positive or negative).
  • this inventions features a method for concentrating and reacting analytes or reactants at any specific micro-location on the device. After the attachment of the specific binding entities, the underlying microelectrode at each micro-location continues to function in a direct current (DC) mode.
  • DC direct current
  • This unique feature allows relatively dilute charged analytes or reactant molecules free in solution to be rapidly transported, concentrated, and reacted in a serial or parallel manner at any specific micro-locations which are maintained at the opposite charge to the analyte or reactant molecules.
  • Specific micro-locations can be protected or shielded by maintaining them at the same charge as the analytes or reactants molecules. This ability to concentrate dilute analyte or reactant molecules at selected micro-locations greatly accelerates the reaction rates at these micro-locations.
  • the microelectrode potential can be reversed to remove non-specific analytes or unreacted molecules from the micro-locations.
  • Specific analytes or reaction products may be released from any micro-location and transported to other locations for further analysis; or stored at other addressable locations; or removed completely from the system.
  • this invention features a method for improving stringency control of nucleic acid hybridization reactions, comprising the steps of:
  • this invention features a method for synthesizing biopolymers at micro-locations.
  • this invention features a method for replicating a master device.
  • this invention features methods for detecting and analyzing reactions that have occurred at the addressed micro-locations using self-addressed microelectronic devices with associated optical, optoelectronic or electronic detection systems or self-addressed microelectronic devices with integrated optical, optoelectronic or electronic detection systems.
  • FIG. 1 is the cross-section of three self-addressable micro-locations fabricated using microlithographic techniques.
  • FIG. 2 is the cross-section of a microlithographically fabricated micro-location.
  • FIG. 3 is a schematic representation of a self-addressable 64 micro-location chip.
  • FIG. 4 shows particular attachment chemistry procedure which allows rapid covalent coupling of specific ligonucleotides to the attachment surface of a microlocation.
  • FIG. 5 is a blown-up schematic diagram of a micromachined 96 micro-locations device.
  • FIG. 6 is the cross-section of a micro-machined device.
  • FIG. 7a and FIG. 7b show the mechanism the device uses to electronically concentrate analyte or reactant molecules at a specific micro-location, FIG. 7a sowing the addressable microlocations in a neutral condition and FIG. 7b showing the addressable microlocations in a charged state.
  • FIGS. 8a, 8b, 8c and 8d show the self-directed assembly of a device with three specific oligonucleotide binding entities (SSO-A, SSO-B, and SSO-C), FIG. 8a showing a first microlocation (ML-1) being addressed, FIG. 8b showing a second microlocation (ML-2) being addressed, FIG. 8c showing a third microlocation (ML-3) being addressed and FIG. 8d showing the three microlocations after being addressed and assembled.
  • SSO-A, SSO-B, and SSO-C three specific oligonucleotide binding entities
  • FIGS. 9a, 9b and 9c show an electronically controlled hybridization process with sample/target DNA being concentrated at micro-locations containing specific DNA capture sequences, FIG. 9a showing specific capture sequences on addressable microlocations, FIG. 9b showing specific and nonspecific DNA adjacent the structure of FIG. 9a, and FIG. 9c showing hybridized material adjacent microlocations ML-1 and ML-3.
  • FIGS. 10a and 10b show an electronically directed serial hybridization process, FIG. 10a showing materials adjacent microlocation ML-3 and FIG. 10b showing materials adjacent microlocations ML-3 and ML-5.
  • FIGS. 11a, 11b and 11c show the electronic stringency control (ESC) of a hybridization process for determining single point mutations, FIG. 11a showing uncharged addressable microlocations, FIG. 11b showing negatively charged microlocations and FIG. 11c showing negatively charged microlocations with material denatured from microlocation ML-3.
  • ESC electronic stringency control
  • FIGS. 12a, 12b, 12c and 12d show a scheme for the detection of hybridized DNA without using labeled DNA probe, i.e., electronically controlled fluorescent dye detection process, FIG. 12a showing uncharged microlocations, FIG. 12b showing negatively charged microlocations, FIG. 12c showing uncharged microlocations with dye and FIG. 12d showing positively charged.
  • FIG. 13a, 13b and 13c show a scheme of electronically controlled replication of devices, FIG. 13a showing negatively charged addressable microlocations, FIG. 13b showing two opposed substrates, one substrate being that of FIG. 13a and the other being a sister device containing an attachment layer, and FIG. 13c showing two substrates, each of which has sequences bound to the microlocations.
  • FIGS. 14a, 14b, 14c, 14d, 14e, and 14f show a scheme of electronically directed combinatorial synthesis of oligonucleotides, FIG. 14a showing addressable microlocations with blocking groups, FIG. 14b showing addressable microlocations with blocking groups in combination with a deblocking group, FIG. 14c showing blocked and deblocked addressable microlocations in the presence of monomer C, FIG. 14d showing addressable microlocations in combination with a deblocking group, FIG. 14e showing deblocked cites on microlocation ML-2 in the presence of monomer A and FIG. 14f showing microlocations with blocking groups on the terminal ends of sequences.
  • the devices and the related methodologies of this invention allow important molecular biology and diagnostic reactions to be carried out under complete electronic control.
  • the basic concept of this invention is a microelectronic device with specially designed addressable microscopic locations. Each micro-location has a derivatized surface for the covalent attachment of specific binding entities (i.e., an attachment layer), a permeation layer, and an underlying direct current (DC) micro-electrode.
  • the device After the initial fabrication of the basic microelectronic structure, the device is able to self-direct the addressing of each specific micro-location with specific binding entities.
  • the self-addressed device is subsequently able to actively carry out multi-step, combinatorial, and multiplex reactions at any of its micro-locations.
  • the device is able to electronically direct and control the rapid movement and concentration of analytes and reactants to or from any of its microlocations.
  • the ability of the device to electronically control the dynamic aspects of various reactions provides a number of new and important advantages and improvements.
  • the concepts and embodiments of this invention are described in three sections.
  • the first section “Design and Fabrication of the Basic Devices,” describes the design of the basic underlying microelectronic device and the fabrication of the device using microlithographic and micromachining techniques.
  • the second section “Self-Directed Addressing of the Devices,” describes the self-addressing and self-assembly of the device, specifically the rapid transport and attachment of specific binding entities to each micro-location.
  • the third section “Applications of the Devices,” describes how the device provides electronic control of various multi-step, combinatorial, and multiplex reactions. This section also describes the various uses and applications of the device.
  • each microlocation must have an underlying functioning DC mode micro-electrode.
  • Other considerations for the design and fabrication of a device include, but are not limited to, materials compatibilities, nature of the specific binding entities and the subsequent reactants and analytes, and the number of micro-locations.
  • a functioning DC mode micro-electrode is meant a micro-electrode biased either positively or negatively, operating in a direct current mode (either continuous or pulse), which can affect or cause the free field electrophoretic transport of charged specific binding entities, reactants, or analytes to or from any location on the device, or in the sample solution.
  • the free field electrophoretic transport of molecules is not dependent on the electric field produced being bounded or confined by dielectrical material.
  • a device can be designed to have as few as two addressable micro-locations or as many as hundreds of thousands of micro-locations.
  • a complex device with a large number of micro-locations is fabricated using microlithography techniques. Fabrication is carried out on silicon or other suitable substrate materials, such as glass, silicon dioxide, plastic, or ceramic materials. These microelectronic "chip" designs would be considered large scale array or multiplex analysis devices.
  • a device with a small number of microlocations would be fabricated using micro-machining techniques.
  • Addressable micro-locations can be of any shape, preferably round, square, or rectangular.
  • the size of an addressable micro-location can be of any size, preferably range from sub-micron ( ⁇ 0.5 ⁇ m) to several centimeters (cm), with 5 ⁇ m to 100 ⁇ m being the most preferred size range for devices fabricated using microlithographic techniques, and 100 ⁇ m to 5 millimeters being the most preferred size range for devices fabricated using the micro-machining techniques.
  • To make micro-locations smaller than the resolution of microlithographic methods would require techniques such as electron beam lithography, ion beam lithography, or molecular beam epitaxy. While microscopic locations are desirable for analytical and diagnostic type applications, larger addressable locations (e.g., larger than 2 mm) are desirable for preparative scale biopolymer synthesis.
  • micro-locations have been created by using microlithographic and/or micro-machining techniques
  • chemical techniques are used to create the specialized attachment and permeation layers which would allow the DC mode micro-electrodes under the micro-locations to: (1) affect or cause the free field electrophoretic transport of specific (charged) binding entities from any location; (2) concentrate and covalently attach the specific binding entities to the specially modified surface of the specific micro-location; and (3) continue to actively function in the DC mode after the attachment of specific binding entities so that other reactants and analytes can be transported to or from the micro-locations.
  • FIG. 1 shows a basic design of self-addressable micro-locations fabricated using microlithographic techniques.
  • the three micro-locations (10) (ML-1, ML-2, ML-3) are formed on the surface of metal sites (12) which have been deposited on an insulator layer/base material.
  • the metal sites (12) serve as the underlying microelectrode structures (10).
  • An insulator material separates the metal sites (12) from each other. Insulator materials include, but are not limited to, silicon dioxide, glass, resist, rubber, plastic, or ceramic materials.
  • FIG. 2 shows the basic features of an individual micro-location (10) formed on a microlithographically produced metal site (12).
  • the addressable micro-location is formed on the metal site (12), and incorporates an oxidation layer (20), a permeation layer (22), an attachment layer (24), and a binding entity layer (26).
  • the metal oxide layer provides a base for the covalent coupling of the permeation layer.
  • the permeation layer provides spacing between the metal surface and the attachment/binding entity layers and allows solvent molecules, small counter-ions, and gases to freely pass to and from the metal surface.
  • the thickness of the permeation layer for microlithographically produced devices can range from approximately 1 nanometers (nm) to 10 microns ( ⁇ m), with 2 nm to 1 ⁇ m being the most preferred.
  • the attachment layer provides a base for the covalent binding of the binding entities.
  • the thickness of the attachment layer for microlithographically produced devices can range from 0.5 nm to 1 ⁇ m, with 1 nm to 200 nm being the most preferred. In some cases, the permeation and attachment layers can be formed from the same material.
  • the specific binding entities are covalently coupled to the attachment layer, and form the specific binding entity layer.
  • the specific binding entity layer is usually a mono-layer of the specific binding molecules. However, in some cases the binding entity layer can have several or even many layers of binding molecules.
  • oligonucleotide binding entities can be attached to one type of micro-location surface without causing a loss of the DC mode function, i.e., the underlying micro-electrode can still cause the rapid free field electrophoretic transport of other analyte molecules to or from the surface to which the oligonucleotide binding entities are attached.
  • large globular protein binding entities e.g., antibodies
  • they may effectively insulate the surface and cause a decrease or a complete loss of the DC mode function.
  • Appropriate modification of the attachment layer would have to be carried out so as to either reduce the number of large binding entities (e.g., large globular proteins) or provide spacing between the binding entities on the surface.
  • the spacing between micro-locations is determined by the ease of fabrication, the requirement for detector resolution between micro-locations, and the number of micro-locations desired on a device.
  • particular spacings between micro-locations, or special arrangement or geometry of the micro-locations is not necessary for device function, in that any combination of microlocations (i.e., underlying micro-electrodes) can operate over the complete device area.
  • the device accomplishes this by attaching the specific binding molecules and subsequent analytes and reactants to the surface of an addressable micro-location. Free field electrophoretic propulsion provides for the rapid and direct transport of any charged molecule between any and all locations on the device.
  • micro-location grouping patterns have to be changed and spacing distances increased proportionally, or multi-layer circuitry can be fabricated into the basic device.
  • a device will contain some un-addressed, or plain micro-locations which serve other functions. These micro-locations can be used to store reagents, to temporarily hold reactants or analytes, and as disposal units for excess reactants, analytes, or other interfering components in samples. Other un-addressed micro-locations can be used in combination with the addressed micro-locations to affect or influence the reactions that are occurring at these specific micro-locations. These micro-locations add to intra-device activity and control. It is also possible for the micro-locations to interact and transport molecules between two separate devices. This provides a mechanism for loading a working device with binding entities or reactants from a storage device, and for copying or replicating a device.
  • FIG. 3 shows a matrix type device containing 64 addressable micro-locations (30).
  • a 64 micro-location device is a convenient design, which fits with standard microelectronic chip packaging components. Such a device is fabricated on a silicon chip substrate approximately 1.5 cm ⁇ 1.5 cm, with a central area approximately 750 ⁇ m ⁇ 750 ⁇ m containing the 64 micro-locations.
  • Each microlocation (32) is approximately 50 ⁇ m square with 50 ⁇ m spacing between neighboring micro-locations.
  • Connective circuitry for each individual underlying micro-electrode runs to an outside perimeter (10 mm ⁇ 10 mm) of metal contact pads (300 ⁇ m square) (34).
  • a raised inner perimeter can be formed between the area with the microlocations and the contact pads, producing a cavity which can hold approximately 2 to 10 microliters ( ⁇ l) of a sample solution.
  • the "chip” can be mounted in a standard quad package, and the chip contact pads (34) wired to the quad package pins. The packaged chip can then be plugged into a microprocessor controlled DC power supply and multimeter apparatus which can control and operate the device
  • the 64 micro-location device (30) shown in FIG. 3 can be fabricated using relatively simple mask design and standard microlithographic techniques.
  • the base substrate material would be a 1 to 2 centimeter square silicon wafer or a chip approximately 0.5 millimeter in thickness.
  • the silicon chip is first overcoated with a 1 to 2 ⁇ m thick silicon dioxide (SiO 2 ) insulation coat, which is applied by plasma enhanced chemical vapor deposition (PECVD).
  • PECVD plasma enhanced chemical vapor deposition
  • a 0.2 to 0.5 ⁇ m metal layer (e.g., aluminum) is deposited by vacuum evaporation.
  • suitable metals for circuitry include gold, silver, tin, copper, platinum, palladium, carbon, and various metal combinations. Special techniques for ensuring proper adhesion to the insulating substrate materials (SiO 2 ) are used with different metals.
  • the chip is next overcoated with a positive photoresist (Shipley, Microposit AZ 1350 J), masked (light field) with the circuitry pattern, exposed and developed.
  • the photosolubilized resist is removed, and the exposed aluminum is etched away.
  • the resist island is now removed, leaving the aluminum circuitry pattern on the chip. This includes an outside perimeter of metal contact pads, the connective circuitry (wires), and the center array of micro-electrodes which serve as the underlying base for the addressable micro-locations.
  • the chip is overcoated first with a 0.2 to 0.4 micron layer of SiO 2 , and then with a 0.1 to 0.2 micron layer of silicon nitride (Si 3 N 4 ).
  • the chip is then covered with positive photoresist, masked for the contact pads and micro-electrode locations, exposed, and developed.
  • Photosolubilized resist is removed, and the SiO 2 and Si 3 N 4 layers are etched away to expose the aluminum contact pads and micro-electrodes.
  • the surrounding island resist is then removed, the connective wiring between the contact pads and the micro-electrodes remains insulated by the SiO 2 and Si 3 N 4 layers.
  • the SiO 2 and Si 3 N 4 layers provide important properties for the functioning of the device.
  • the second SiO 2 layer has better contact and improved sealing with the aluminum circuitry. It is also possible to use resist materials to insulate and seal. This prevents undermining of the circuitry due to electrolysis effects when the micro-electrodes are operating.
  • the final surface layer coating of Si 3 N 4 is used because it has much less reactivity with the subsequent reagents used to modify the micro-electrode surfaces for the attachment of specific binding entities.
  • micro-electrode locations on the device are ready to be modified with a specialized permeation and attachment layer.
  • the objective is to create on the micro-electrode an intermediate permeation layer with selective diffusion properties and an attachment surface layer with optimal binding properties.
  • the attachment layer should have from 10 5 to 10 7 functionalized locations per square micron ( ⁇ m 2 ) for the optimal attachment of specific binding entities.
  • the attachment of specific binding entities must not overcoat or insulate the surface so as to prevent the underlying micro-electrode from functioning.
  • a functional device requires some fraction (-5% to 25%) of the actual metal micro-electrode surface to remain accessible to solvent (H 2 O) molecules, and to allow the diffusion of counter-ions (e.g., Na + and Cl - ) and electrolysis gases (e.g., O 2 and H 2 ) to occur.
  • counter-ions e.g., Na + and Cl -
  • electrolysis gases e.g., O 2 and H 2
  • the intermediate permeation layer must also allow diffusion to occur. Additionally, the permeation layer should have a pore limit property which inhibits or impedes the larger binding entities, reactants, and analytes from physical contact with the micro-electrode surface. The permeation layer keeps the active microelectrode surface physically distinct from the binding entity layer of the micro-location.
  • this design allows the electrolysis reactions required for electrophoretic transport to occur on micro-electrode surface, but avoids adverse electrochemical effects to the binding entities, reactants, and analytes.
  • APS aminopropyltriethoxy silane
  • APS reacts readily with the oxide and/or hydroxyl groups on metal and silicon surfaces.
  • APS provides a combined permeation layer and attachment layer, with primary amine groups for the subsequent covalent coupling of binding entities.
  • APS produces a relatively high level of functionalization (i.e., a large number of primary amine groups) on slightly oxidized aluminum surfaces, an intermediate level of functionalization on SiO 2 surfaces, and very limited functionalization of Si 3 N 4 surfaces.
  • the APS reaction is carried out by treating the whole device (e.g., a chip) surface for 30 minutes with a 10% solution of APS in toluene at 50° C. The chip is then washed in toluene, ethanol, and then dried for one hour at 50° C.
  • the micro-electrode metal surface is functionalized with a large number of primary amine groups (10 5 to 10 6 per square micron). Binding entities can now be covalently bound to the derivatized micro-electrode surface.
  • FIG. 4 shows the mechanism for the attachment of 3'-terminal aldehyde derivatized oligonucleotides (40) to an APS functionalized surface (42). While this represents one of the preferred approaches, a variety of other attachment reactions are possible for both the covalent and non-covalent attachment of many types of binding entities.
  • micro-machining techniques e.g., drilling, milling, etc.
  • non-lithographic techniques to fabricate devices.
  • these devices have relatively larger micro-locations (>100 microns) than those produced by microlithography.
  • These devices could be used for analytical applications, as well as for preparative type applications, such as biopolymer synthesis.
  • Large addressable locations could be fabricated in three dimensional formats (e.g., tubes or cylinders) in order to carry a large amount of binding entities.
  • Such devices could be fabricated using a variety of materials, including, but not limited to, plastic, rubber, silicon, glass (e.g., microchannelled, microcapillary, etc.), or ceramics.
  • connective circuitry and larger electrode structures can be printed onto materials using standard circuit board printing techniques known to those skilled in the art.
  • FIG. 5 is a schematic of a representative 96 microlocation device.
  • This micro-location device is fabricated from a suitable material stock (2 cm ⁇ 4 cm ⁇ 1 cm), by drilling 96 proportionately spaced holes (1 mm in diameter) through the material.
  • An electrode circuit board (52) is formed on a thin sheet of plastic material stock, which fit precisely over the top of the micro-location component (54).
  • the underside of the circuit board contains the individual wires (printed circuit) to each micro-location (55).
  • Short platinum electrode structures ( ⁇ 3-4 mm) (62) are designed to extended down into the individual micro-location chambers (57).
  • the printed circuit wiring is coated with a suitable water-proof insulating material.
  • the printed circuit wiring converges to a socket, which allows connection to a multiplex switch controller (56) and DC power supply (58).
  • the device is partially immersed and operates in a common buffer reservoir (59).
  • the permeation and attachment layers are formed directly on the underlying metal micro-electrode.
  • the permeation and attachment layers are physically separated from their individual metal electrode structure (62) by a buffer solution in the individual chamber or reservoir (57) (see FIG. 6).
  • the permeation and attachment layers can be formed using functionalized hydrophilic gels, membranes, or other suitable porous materials.
  • the thickness of the combined permeation and attachment layers ranges from 10 ⁇ m to 10 mm.
  • a modified hydrophilic gel of 26% to 35% polyacrylamide (with 0.1% polylysine) can be used to partially fill ( ⁇ 0.5 mm) each of the individual micro-location chambers in the device.
  • This concentration of gel forms an ideal permeation layer with a pore limit of from 2 nm to 3 nm.
  • the polylysine incorporated into the gel provides primary amine functional groups for the subsequent attachment of specific binding entities.
  • This type of gel permeation layer allows the electrodes to function actively in the DC mode. When the electrode is activated, the gel permeation layer allows small counter-ions to pass through it, but the larger specific binding entity molecules are concentrated on the outer surface. Here they become covalently bonded to the outer layer of primary amines, which effectively becomes the attachment layer.
  • the disclosed devices are able to electronically self-address each micro-location with a specific binding entity.
  • the device itself directly affects or causes the transport and attachment of specific binding entities to specific micro-locations.
  • the device self-assembles itself in the sense that no outside process, mechanism, or equipment is needed to physically direct, position, or place a specific binding entity at a specific microlocation. This self-addressing process is both rapid and specific, and can be carried out in either a serial or parallel manner.
  • a device can be serially addressed with specific binding entities by maintaining the selected microlocation in a DC mode and at the opposite charge (potential) to that of a specific binding entity. All other micro-locations are maintained at the same charge as the specific binding entity. In cases where the binding entity is not in excess of the attachment sites on the micro-location, it is necessary to activate only one other micro-electrode to affect the electrophoretic transport to the specific micro-location.
  • the specific binding entity is rapidly transported (in a few seconds, or preferably less than a second) through the solution, and concentrated directly at the specific micro-location where it immediately becomes covalently bonded to the special surface.
  • the ability to electronically concentrate reactants or analytes (70) on a specific micro-location (72) is shown in FIGS. 7a and 7b.
  • FIGS. 8a through 8d shows the serial process for addressing specific micro-locations (81, 83, 85) with specific oligonucleotide binding entities (82, 84, 86).
  • the parallel process for addressing micro-locations simply involves simultaneously activating a large number (particular group or line) of micro-electrodes so that the same specific binding entity is transported, concentrated, and reacted with more than one specific micro-locations.
  • the devices of this invention are able to electronically provide active or dynamic control over a number of important reaction parameters.
  • This electronic control leads to significant improvements in reaction rates, specificities, and sensitivities.
  • the improvements in these reaction parameters come from the ability of the device to electronically control and affect: (1) the rapid transport of reactants or analytes to a specific micro-location containing attached specific binding entities; (2) improvement in reaction rates due to the concentrated reactants or analytes reacting with the specific binding entities at that specific micro-location; and (3) the rapid and selective removal of un-reacted and non-specifically bound components from that micro-location.
  • the self-addressed devices of this invention are able to rapidly carry out a variety of micro-formatted multistep and/or multiplex reactions and procedures; which include, but are not limited to:
  • molecular biology reaction procedures e.g., restriction enzyme reactions and analysis, ligase reactions, kinasing reactions, and amplification procedures;
  • diagnostic assays e.g., hybridization analysis, gene analysis, fingerprinting, and immuno-diagnostics
  • biomolecular conjugation procedures i.e. the covalent and non-covalent labeling of nucleic acids, enzymes, proteins, or antibodies with reporter groups
  • biopolymer synthesis procedures e.g., combinatorial synthesis of oligonucleotides or peptides
  • water soluble synthetic polymer synthesis e.g., carbohydrates or linear polyacrylates
  • Nucleic acid hybridizations are used as examples of this invention because they characterize the most difficult multi-step and multiplex reactions.
  • the device and methods allow nucleic acid hybridization to be carried out in a variety of conventional and new formats.
  • the ability of the device to electronically control reaction parameters greatly improves nucleic acid hybridization analysis, particularly the ability of the device to provide electronic stringency control (ESC).
  • ESC electronic stringency control
  • nucleic acid hybridization hybridization between all natural and synthetic forms and derivatives of nucleic acids, including: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), polynucleotides and oligonucleotides.
  • a device for DNA hybridization analysis is designed, fabricated, and used in the following manner.
  • Arrays of micro-locations are first fabricated using microlithographic techniques. The number of addressable micro-locations on an array depends on the final use.
  • the device is rapidly self-addressed in a serial manner with a group of specific oligonucleotides.
  • the specific oligonucleotides are 3'-terminal aldehyde functionalized oligonucleotides (in the range of 6-mer to 100-mer).
  • the aldehyde functional group allows for covalent attachment to the specific microlocation attachment surface (see FIG. 4).
  • This group of specific oligonucleotides can be readily synthesized on a conventional DNA synthesizer using conventional techniques.
  • each specific oligonucleotide is initiated from a ribonucleotide controlled pore glass (CPG) support.
  • CPG ribonucleotide controlled pore glass
  • the 3'-terminal position contains a ribonucleotide, which is then easily converted after synthesis and purification to a terminal dialdehyde derivative by periodate oxidation.
  • the aldehyde containing oligonucleotides (40) will react readily with the primary amine functional groups on the surface of micro-locations by a Schiff's base reaction process.
  • the electronic addressing of the device with specific oligonucleotides is shown in FIG. 8.
  • the addressing of the first specific micro-location (ML-1) (81) with its specific sequence oligonucleotide (SSO-1) (82) is accomplished by maintaining the specific microelectrode (ML-1) at a positive DC potential, while all other microelectrodes are maintained at a negative potential (FIG. 8(A)).
  • the aldehyde functionalized specific sequence (SSO-1) in aqueous buffered solution is free field electrophoresed to the ML-1 address, where it concentrates (>10 6 fold) and immediately becomes covalently bound to the surface of ML-1 (81).
  • All other microelectrodes are maintained negative, and remain protected or shielded from reacting with SSO-1 sequence (82).
  • the ML-1 potential is then reversed to negative (-) to electrophores any unreacted SSO-1 to a disposal system.
  • the cycle is repeated, SSO-2 (84) ⁇ ML-2 (83), SS0-3 (86) ⁇ ML-3 (85), SSO-n ⁇ ML-n until all the desired micro-locations are addressed with their specific DNA sequences (FIG. 8(D)).
  • binding entities such as specific oligonucleotides
  • This supply device would hold a large quantity of binding entities or reagents and would be used to load analytical devices. Binding entities would be electronically transported between the two devices. Such a process eliminates the need for physical manipulations, such as pipetting, in addressing a device with binding entities.
  • Yet another method for addressing the device is to carry out the combinatorial synthesis of the specific oligonucleotides at the specific micro-locations. Combinatorial synthesis is described in a later section.
  • the micro-locations on the array device remain as independent working direct current (DC) electrodes. This is possible because the attachment to the electrode surface is carried out in such a manner that the underlying micro-electrode does not become chemically or physically insulated. Each micro-electrode can still produce the strong direct currents necessary for the free field electrophoretic transport of other charged DNA molecules to and from the micro-location surface.
  • the DNA array device provides complete electronic control over all aspects of the DNA hybridization and any other subsequent reactions.
  • each addressable micro-location has a specific capture sequence (90).
  • a sample solution containing target DNA (92) is applied to the device. All the micro-locations are activated and the sample DNA is concentrated at the microlocations (FIG. 9(B)).
  • Target DNA molecules from the dilute solution become highly concentrated at the microlocations, allowing very rapid hybridization to the specific complementary DNA sequences on the surface.
  • Reversal of the micro-electrode potential repels all unhybridized DNA from the micro-locations, while the target DNA remains hybridized (FIG. 9(C)).
  • reporter probes are hybridized in subsequent steps to detect hybridized complexes.
  • the electronic control of the hybridization process significantly improves the subsequent detection of the target DNA molecules by enhancing the overall hybridization efficiency and by removing non-specific DNA from the micro-location areas. It is expected that 10,000 to 100,000 copies of target sequences in un-amplified genomic DNA will be detectable. Hybridization reactions of this type can be carried out in a matter of minutes, with minimal outside manipulations. Extensive washing is not necessary.
  • FIGS. 10a and 10b shows an improved version of this serial hybridization format.
  • micro-locations 101-107 are addressed with different capture DNAs.
  • sequence specific oligonucleotides 108,109.
  • the microlocations are sequentially biased positive to transport molecules to itself and then biased negative to transport molecules to the next micro-location. Specifically hybridized DNA will remain at the micro-location regardless of electrode potential.
  • the sequence specific oligonucleotides can be labeled with a suitable reporter group such as a fluorophore.
  • the disclosed device is able to provide electronic stringency control.
  • Stringency control is necessary for hybridization specificity, and is particularly important for resolving one base mismatches in point mutations.
  • FIGS. 11a through 11c shows how electronic stringency control can be used for improving hybridization specificity for one base mismatch analysis.
  • the electronic stringency control can also be applied to multiple-base mismatch analysis.
  • the disclosed device provides independent stringency control to each specific hybridization reaction occurring on the device.
  • a conventional or passive array format it is impossible to achieve optimal stringency for all the hybridization events which are occurring in the same hybridization solution.
  • the active array devices of this invention are able to provide different electronic stringency to hybridizations at different micro-locations, even though they are occurring in the same bulk hybridization solution. This attribute overcomes a major limitation to conventional matrix hybridization formats, sequencing by hybridization (SBH) formats, and other multiplex analyses.
  • the ability to provide electronic stringency control to hybridizations also provides mechanisms for detecting DNA hybridization without reporter group labeled DNA probe. It provides a way to carry out a more direct detection of the hybridization process itself.
  • a fluorescent dye detection process is shown in FIGS. 12a through 12d and described in Examples 4 and 6. Direct detection of DNA hybrids can be achieved by using DNA binding dyes such as ethidium bromide. The dye binds to both double-stranded and single-stranded DNA but with a greater affinity for the former. In FIG. 12(B) positively charged dye (122) is transported to negatively biased micro-locations. The dye binds to both hybridized (120) and unhybridized (121) DNA sequences (FIG. 12c).
  • the dye molecules bound to unhybridized micro-locations is selectively removed.
  • the amount of power applied does not adversely affect the DNA hybrids.
  • the hybridized DNAs with associated dye molecules are then fluorescently detected using associated or integrated optical systems.
  • FIGS. 13a through 13c A master device containing micro-locations which have been addressed with specific binding sequences is hybridized with respective complementary DNA sequences (130). These complementary sequences are activated and thus capable of covalent binding to the micro-location attachment layer.
  • An unaddressed sister device (132) containing an attachment layer is aligned with the hybridized master device (FIG. 13(B)).
  • the master device micro-locations are biased negative and the sister device micro-locations are biased positive.
  • the DNA hybrids are denatured and are transported to the sister device, where the activated DNA sequence binds covalently to the micro-location (FIG. 13(C)).
  • the process can be performed in parallel or in series, depending on the device geometry so that crosstalk between the micro-locations is minimized.
  • the hybrids can be denatured by applying a sufficient negative potential or by using a positively charged chaotropic agent or denaturant.
  • an epifluorescent type microscopic detection system for the analysis of the binding reactions.
  • the sensitivity of the system depends on the associated imaging detector element (CCD, ICCD, Microchannel Plate) or photon counting PMT system.
  • CCD imaging detector element
  • ICCD Integrated Cellular Cell Sorting Device
  • APD avalanche photodiode
  • Another alternative is to integrate optoelectronic or microelectronics detection in the device.
  • the devices of this invention are also capable of carrying out combinatorial synthesis of biopolymers such as oligonucleotides and peptides. Such a process allows self-directed synthesis to occur without the need for any outside direction or influence.
  • This combinatorial synthesis allows very large numbers of sequences to be synthesized on a device.
  • the basic concept for combinatorial synthesis involves the use of the device to transport, concentrate, and react monomers, coupling reagents, or deblocking reagents at the addressable micro-locations. The concept capitalizes on the ability of the device to protect certain locations from the effects of nearby reagents. Also important to the .concept is the identification of selective steps in these chemical synthesis processes where one or more of the reactants has either a net positive or negative charge, or to create such suitable reagents for these processes.
  • FIGS. 14a through 14f One method for combinatorial oligonucleotide synthesis is shown in FIGS. 14a through 14f.
  • This method begins with a set of selectively addressable micro-locations (140) whose surfaces have been derivatized with blocked primary amine (X-NH-) groups (142).
  • the initial step in the process involves selective deblocking of electrodes using a charged deblocking reagent (144). In this case, the reagent would carry a positive (+) charge.
  • the process is carried out by applying a negative potential to those electrodes being de-blocked, and a positive potential to those which are to remain protected (FIG. 14(B)).
  • Application of positive and negative potentials to selective electrodes causes the charged reagents to be concentrated at those micro-locations being de-blocked, and excludes the reagents from the other electrode surfaces.
  • the second step chemical coupling of the first base, in this case cytosine, to the deblocked microlocations is carried out by simply exposing the system to the phosphoramidite reagent (x-C) (146).
  • the (C) nucleotide couples to de-blocked micro-location surfaces, but not to any of the blocked electrode surfaces (FIG. 14(C) and (D)). At this point normal phosphoramide chemistry is carried out until the next de-blocking step.
  • the above example represents one possible approach for the synthesis of nucleic acids.
  • Another approach involves a complete water soluble DNA synthesis.
  • charged water soluble coupling agents such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCA)
  • EDCA 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • EDCA 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • This approach would have significant advantages over present organic solvent based methods which require extensive blocking of the base moieties. Water soluble synthesis would be less expensive and eliminate the use of many toxic substances used in the present organic solvent based processes.
  • a third approach involves the use of charged monomers.
  • Synthetic DNA probes were made using conventional phosphoramidite chemistry on Applied Biosystems automated DNA synthesizers. Oligomers were designed to contain either a 5' amino or a 3' ribonucleoside terminus. The 5' functionality was incorporated by using the ABI Aminolink reagent and the 3' functionality was introduced by initiating synthesis from an RNA CPG support. The 3' ribonucleotide terminus can be converted to a terminal dialdehyde by the periodate oxidation method which can react with primary amines to form a Schiff's base. Reaction conditions were as follows: Dissolve 20-30 O.D. oligomer in water to a final concentration of 1 OD/ ⁇ l.
  • oligomers contain 3' ribonucleoside termini (U): ##STR1##
  • Oligomers containing 5' amine groups were generally reacted with fluorophores, such as Texas Red (TR, ex. 590 nm, em. 610 nm). Sulfonyl chlorides are very reactive towards primary amines forming a stable sulfonamide linkage.
  • Texas Red-DNA conjugates were made as follows: Texas Red sulfonyl chloride (Molecular Probes) was dissolved in dimethyl formamide (DMF) to a final concentration of 50 mg/ml (80 mM). Oligomer was dissolved in 0.4M sodium bicarbonate, pH 9.0-9.1, to a final concentration of 1 O.D./ ⁇ l (5.4 mM for a 21-mer).
  • EXAMPLE 2 Electronically Addressable Micro-locations on a Microfabricated Device--Polylysine Method
  • Microelectrodes were fabricated from microcapillary tubes (0.2 mm ⁇ 5 mm).
  • the microcapillaries were filled with 18-26% polyacrylamide containing 0.1-1.0% polylysine and allowed to polymerize. The excess capillary was scored and removed to prevent air bubbles from being trapped within the tubes and to standardize the tube length.
  • Capillaries were mounted in a manner such that they shared a common upper buffer reservoir and had individual lower buffer reservoirs. Each lower buffer reservoir contained a platinum wire electrode.
  • the top surface of the microcapillary in the upper reservoir was considered to be the addressable microlocation.
  • Upper and lower reservoirs were filled with 0.1M sodium phosphate, pH 7.4 and prerun for 10' at 0.05 mA constant using a BioRad 500/1000 power supply. Pipette 2 ⁇ l (0.1 O.D.) periodate oxidized ET12R into the upper reservoir while the power is on and electrophorese for 2-5 minutes at constant current. Reverse polarity so that the test capillary is now biased negative and electrophorese an additional 2-5 minutes. Unbound DNA is repulsed while the covalently attached DNA remains.
  • EXAMPLE 3 Electronically Addressable Micro-locations on a Microfabricated Device--Succinimidyl Acrylate Method
  • This example describes an alternative attachment chemistry which covalently binds the 5' terminus of the oligomer.
  • Capillaries were fabricated as described above except that 1% succinimidyl acrylate (Molecular Probes) was substitute for the polylysine. The capillaries were made fresh because the succinimidyl ester reacts with primary amines and is labile, especially above pH 8.0. The capillaries were mounted as described above and the reservoirs were filled with 0.1M sodium phosphate, pH 7.4. Prerun the capillaries for 10 minutes at 0.05 mA. Pipette 2 ⁇ l ET10AL (0.1 O.D.), which contains a 5' amino terminus, into the upper reservoir while the power is on and electrophorese for 2-5 minutes. Reverse polarity so that the test capillary is now biased negative and electrophorese an additional 2-5 minutes. Unbound DNA is repulsed while the covalently attached DNA remains.
  • succinimidyl acrylate Molecular Probes
  • DNA dyes such as ethidium bromide (EB) become fluorescent when intercalated into DNA.
  • the fluorescence and binding affinity is greater when the DNA is double stranded than single stranded.
  • EB was added to the solution ( ⁇ 0.05 mM EB final concentration) and the test capillary was biased negative because EB is positively charged.
  • the capillaries were observed by epifluorescence at 550 nm excitation and 600+ nm emission. Both the hybridized and unhybridized micro-locations showed red fluorescence (from EB).
  • the capillaries were re-mounted biased positive to repulse EB. Maintain constant current at 0.05 mA for 0.03 Volt-Hours.
  • Aluminum (Al) and gold (Au) wire (0.25 mm, Aldrich) was reacted with 10% 3-aminopropyltriethoxysilane (APS) in toluene.
  • APS 3-aminopropyltriethoxysilane
  • the gold wire was subjected to electrolysis in 5 ⁇ SSC solution to form an oxide layer.
  • the metal wire can be oxidized by a perchloric acid bath.
  • the APS reaction was performed as follows: Wires were cut to 3 inches and placed in a glass dish. Toluene was added to completely cover the wires and the temperature was brought to 50°-60° C. on a heat plate. APS was added to a final concentration of 10%. Mix solution and continue the reaction for 30 minutes. Rinse 3 times with copious volumes of toluene, then rinse 3 times with copious volumes of alcohol and dry in 50° C. oven. The APS treated wire can then be reacted with an aldehyde to form a Schiff's base. Binding entity ET12R was periodate oxidized as described elsewhere. The electrodes were placed in a reservoir of degassed water. Power was applied at 0.05 mA constant for about 30 seconds.
  • Activated ET12R was immediately added. Power was applied, the liquid was aspirated and fresh water was added and then aspirated again.
  • the test (biased positive) and reference electrodes were placed in Hybridization Buffer (HB, 5 ⁇ SSC, 0.1% SDS) containing fluorescent labeled complement DNA, ET10-TR. After 2 minutes the electrodes were washed three times for one minute each in Wash Buffer (1 ⁇ SSC, 0.1% SDS) and observed by fluorescence (ex. 590 nm, em. 610 nm).
  • Results demonstrate that ET12R was specifically coupled to the treated metal surfaces.
  • the test electrode was fluorescent while the reference electrode was not.
  • Nonspecific adsorption of the DNA to the metal was prevented by the presence of SDS in the Hybridization Buffer. Attachment to gold substrates by electrolysis and subsequent APS treatment was effective. Signal obtained was significantly stronger than observed with non-oxidized gold. More importantly, this example showed that the metal surfaces could be chemically functionalized and derivatized with a binding entity and not become insulated from the solution.
  • the APS method represents one of many available chemistries to form DNA-metal conjugates.
  • DNA-aluminum electrode substrates were prepared and hybridized as described in Example 5.
  • a hybridized and an unhybridized DNA-Al electrode were processed with an underivatized Al wire as the reference.
  • EB was added to the solution and the test DNA electrodes were biased negative to attract the dye.
  • the solution was aspirated and fresh buffer was added. The metal surfaces were examined under the microscope.
  • a radial array of 6 addressable 250 ⁇ m capillary locations was micro-machined.
  • the device has a common upper reservoir and separate lower reservoirs such that the potential at each micro-location is individually addressable.
  • a unique oligomer binding entity is localized and coupled to a specific capillary micro-location by the methods described elsewhere.
  • the test micro-location has a positive potential while the other micro-locations have negative potentials to prevent nonspecific interactions.
  • the array is washed and then hybridized with a complementary fluorescently labeled DNA probe.
  • the array is washed to remove excess probe and then observed under an epifluorescent microscope. Only the specifically addressed micro-location will be fluorescent. The process may be repeated with another binding entity at another location and verified by hybridization with a probe labeled with another fluorescent moiety.
  • DNA sequences are specifically located to predetermined positions with negligible crosstalk with the other locations. This enables the fabrication of micromatrices with several to hundreds of unique sequences at predetermined locales.
  • the APS process involves the entire chip. Selectivity of the functionalization process was dependent on the reactivity of the chip surfaces. In order to reduce functionalization and subsequent DNA attachment of the areas surrounding the micro-locations, a material that is less reactive to APS than SiO 2 or metal oxide is needed. Photoresists and silicon nitride were tried. The different topcoats were applied to silicon dioxide chips. The chips were examined by epifluorescence and the then treated with APS followed by covalent attachment of periodate oxidized polyA RNA sequences (Sigma, MW 100,000). The chips were hybridized with 200 nM solution of Texas Red labeled 20-mer (T2-TR) in Hybridization Buffer, for 5 minutes at 37° C. The chips were washed 3 times in WB and once in 1 ⁇ SSC. The chips were examined by fluorescence at 590 nm excitation and 610 nm emission.
  • Silicon nitride was chosen because it had much less reactivity to APS relative to silicon dioxide and was not inherently fluorescent like the photoresist tested. Other methods such as UV burnout of the background areas are also possible.
  • a finished matrix chip was visually examined using a Probe Test Station (Micromanipulator Model 6000) fitted with a B & L microscope and a CCD camera. The chip was tested for continuity between the test pads and the outer contact pads. This was done by contacting the pads with the manipulator probe tips which were connected to a multimeter. Continuity ensures that the pads have been etched down to the metal surface. The pads were then checked for stability in electrolytic environments. The metal wires were rated to handle up to 1 mA under normal dry conditions. However, reaction to a wet environment was unknown. A drop (1-5 ⁇ l) of buffered solution (1 ⁇ SSC) was pipetted onto the 8 ⁇ 8 matrix. Surface tension keeps the liquid in place leaving the outer contact pad area dry. A probe tip was contacted to a contact pad and another probe tip was contacted with the liquid. The current was incremented up to 50 nA at max voltage of 50 V using a HP 6625A power supply and HP3458A digital multimeter.
  • the initial fabrication consisted of the silicon substrate, a silicon dioxide insulating layer, aluminum deposition and patterning, and a silicon nitride topcoat. These chips were not very stable in wet environments because the metal/nitride interface was physical in nature and electrolysis would undermine the nitride layer. This would result in the pads being electrically shorted. Furthermore, silicon nitride and Al have different expansion coefficients such that the nitride layer would crack when current was applied. This would allow solution to contact the metal directly, again resulting in electrolysis which would further undermine the layer.
  • the second fabrication process included a silicon dioxide insulating layer between the aluminum metal and silicon nitride layers. Silicon dioxide and Al have more compatible physical properties and form a better chemical interface to provide a more stabile and robust chip.
  • a matrix chip was functionalized with APS reagent as described in Example 5.
  • the chip was then treated with periodate oxidized polyA RNA (Sigma, average MW 100,000).
  • the chip was washed in WB to remove excess and unbound RNA. This process coated the entire chip with the capture sequence with a higher density at the exposed metal surfaces than at the nitride covered areas.
  • the chip was hybridized with a 200 nM solution of T2-TR in HB for 5 minutes at 37° C. and then washed 3 times in WB and once in 1 ⁇ SSC for one minute each at ambient temperature.
  • the chip was examined by fluorescence at 590 nm excitation and 610 nm emission.
  • the opened metal areas were brightly fluorescent and had the shape of the pads. Low fluorescent intensities and/or irregular borders would suggest that the pads were not completely opened. Additional plasma etch times would be recommended.
  • Active hybridization was performed by using a chip from Example 8c and biasing one micro-location positive. This was done by using the switch box which would also automatically bias the remaining micro-locations negative or by using an external solution electrode. Three microliters of water was deposited on the matrix pads only. A current, ⁇ 1-5 nA, was applied for several seconds and 0.1 pmole of T2-TR was added to the solution. The liquid was removed and the chip was dried and examined by fluorescence at Texas Red wavelengths (ex. 590 nm, em. 610 nm).
  • the fluorescence DNA at one micro-location can be translocated to another micro-location by biasing the initial location negative and the destination positive.
  • Binding entity CP-1 was activated by periodate oxidation method. Four micro-locations were biased positive in the matrix and the remainder were biased negative. Two microliters of water was deposited on the matrix and a current was applied. Binding entity, CP-1, was added and allowed to concentrate at the designated locations. The liquid was removed, the chip was rinsed briefly with water and two microliters of water was deposited on the chip. Again, current was applied for several seconds and 0.1 pmole of T2-TR was added. The liquid was removed after a short time and the entire chip was washed in WB, 3 times. The chip was dried and examined for fluorescence.
  • Results indicate that the positively biased microlocations were fluorescent.
  • This example demonstrates the selective addressing of micro-locations with a specific binding entity, the localization and covalent coupling of sequences to the micro-locations, and the specific hybridization of complementary target sequences to the derivatized micro-locations.
  • DNA binding entities with 3' ribonucleoside termini are synthesized which are specific for the polymorphisms of HLA gene dQa.
  • the binding entities are activated by periodate oxidation as described above.
  • the reverse complements are also synthesized with 5' amino termini and are conjugated with fluorophores, such as Texas Red, Rhodamine or Bodipy dyes, as described elsewhere.
  • the micro-locations are functionalized with primary amines by treatment with APS, as described elsewhere. A couple microliters of solution are placed over the matrix but leaving the contact pads dry. A specific micro-location is addressed by biasing that micro-location positive, the periodate oxidized DNA oligomer is added, ⁇ 0.1 pmole, and is translocated and covalently coupled to that location.
  • the polarity is reversed and the unbound binding entity molecules are removed. This is repeated for another binding entity at another addressed micro-location until all the unique binding entities are bound to the chip.
  • the chip is then hybridized to individual fluorescently labeled complement sequences to determine the specificity of the coupling reaction as well as en masse to visualize all addressed micro-locations at once.
  • the addressed microlocations are hybridized with unlabeled reverse complements or genomic DNA. Detection is via the fluorescent dye detection assay as described elsewhere.
  • Results will demonstrate that micro-locations are specifically addressed with unique binding entities. Nonspecific binding to negatively biased micro-locations will be negligible.
  • the device and associated binding entity chemistry is stable under denaturation conditions, thus making the addressed and fabricated device reusable.
  • Alternative methods for denaturing the hybrids would be to increase the current and/or increase the time it is applied.
  • Microelectrodes are fabricated from microcapillary tubes as described elsewhere. Binding entities Ras-G is periodate oxidized and covalently bound to the addressed micro-location. Ras-G micro-location is then hybridized with Ras-1-TR which is the perfect match, Ras-2-TR which is a one base pair mismatch (G-A) or Ras-3-TR which is a two base pair mismatch (G-A and G-T). The micro-locations are examined fluorescently to verify whether complementary sequences are hybridized and to what extent. The microcapillaries are re-mounted and subjected to controlled time at constant current until the mismatched hybrids are removed without significantly affecting the perfectly matched hybrids.

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US08/146,504 1991-11-07 1993-11-01 Active programmable electronic devices for molecular biological analysis and diagnostics Expired - Lifetime US5605662A (en)

Priority Applications (74)

Application Number Priority Date Filing Date Title
US08/146,504 US5605662A (en) 1993-11-01 1993-11-01 Active programmable electronic devices for molecular biological analysis and diagnostics
US08/271,882 US6017696A (en) 1993-11-01 1994-07-07 Methods for electronic stringency control for molecular biological analysis and diagnostics
US08/304,657 US5632957A (en) 1993-11-01 1994-09-09 Molecular biological diagnostic systems including electrodes
CA002175483A CA2175483C (en) 1993-11-01 1994-10-26 Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics
CNA2006101003286A CN1920055A (zh) 1993-11-01 1994-10-26 可自我寻址自我组装的用于分子生物学分析和诊断的微电子系统及装置
CA002477138A CA2477138C (en) 1993-11-01 1994-10-26 Molecular biological analysis and diagnostics using microelectronic systems
EP01106838A EP1120155A3 (en) 1993-11-01 1994-10-26 Method for combinatorial synthesis of a biopolymer
AT95900430T ATE211259T1 (de) 1993-11-01 1994-10-26 Sich selbst adressierende und zusammenfügende mikroelektronische systeme und vorrichtungen für molekularbiologische analysen und diagnosen
NZ500373A NZ500373A (en) 1993-11-01 1994-10-26 Microelectronic device with electrodes with permeation layers for electrolytic transport of species
PCT/US1994/012270 WO1995012808A1 (en) 1993-11-01 1994-10-26 Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics
ES95900430T ES2170133T3 (es) 1993-11-01 1994-10-26 Dispositivos y sistemas microelectronicos autoensamblables auto-dirigibles para diagnostico y analisis en biologia molecular.
EP01106841A EP1120469A3 (en) 1993-11-01 1994-10-26 Method for actively transporting DNA
PT95900430T PT727045E (pt) 1993-11-01 1994-10-26 Dispositivos e sistemas microelectronicos auto-enderecaveis e auto-configuraveis para diagnostico e analise biologica molecular
JP7513281A JPH09504910A (ja) 1993-11-01 1994-10-26 分子生物学的分析および診断用の自己アドレス可能、自己組立て小型電子システムおよびデバイス
BR9407952A BR9407952A (pt) 1993-11-01 1994-10-26 Sistemas e dispositivos microeletrônicos auto-endereçáveis auto-montável para análise e diagnósticos biológicos moleculares
CA002504343A CA2504343A1 (en) 1993-11-01 1994-10-26 Method for electronic assembly of nanostructures
NZ330036A NZ330036A (en) 1993-11-01 1994-10-26 Electronic multielectrode devices for molecular biology analysis and synthesis
NZ330037A NZ330037A (en) 1993-11-01 1994-10-26 Electronic DNA synthesis techniques
DE69429535T DE69429535T2 (de) 1993-11-01 1994-10-26 Sich selbst adressierende und zusammenfügende mikroelektronische systeme und vorrichtungen für molekularbiologische analysen und diagnosen
AU81257/94A AU692800B2 (en) 1993-11-01 1994-10-26 Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis nad diagnostics
CNA2004100040679A CN1558207A (zh) 1993-11-01 1994-10-26 可自我寻址自我组装的用于分子生物学分析和诊断的微电子系统及装置
CNB941947599A CN1142821C (zh) 1993-11-01 1994-10-26 可自我寻址自我组装的用于分子生物学分析和诊断的微电子系统及装置
EP01106840A EP1120156A3 (en) 1993-11-01 1994-10-26 Method of electronically controlled hybridization of DNA
EP95900430A EP0727045B1 (en) 1993-11-01 1994-10-26 Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics
EP01106846A EP1120157A3 (en) 1993-11-01 1994-10-26 Self-addressable electronic device
NZ275962A NZ275962A (en) 1993-11-01 1994-10-26 Microelectronic device for molecular biology analysis and diagnostic reactions
DK95900430T DK0727045T3 (da) 1993-11-01 1994-10-26 Selvadresserbare, selvsamlende, mikroelektroniske systemer og indretninger til molekylærbiologisk analyse og diagnostik
US08/534,454 US5849486A (en) 1993-11-01 1995-09-27 Methods for hybridization analysis utilizing electrically controlled hybridization
FI961843A FI961843A (sv) 1993-11-01 1996-04-30 Självdresserbara, självhopsättbara mikroelektroniska system och anordningar för molekylbiologisk analys och diagnostik
US08/725,976 US5929208A (en) 1993-11-01 1996-10-04 Methods for electronic synthesis of polymers
US08/726,278 US6238624B1 (en) 1993-11-01 1996-10-04 Methods for transport in molecular biological analysis and diagnostics
US08/753,962 US6287517B1 (en) 1993-11-01 1996-12-04 Laminated assembly for active bioelectronic devices
US08/760,933 US6652808B1 (en) 1991-11-07 1996-12-06 Methods for the electronic assembly and fabrication of devices
US08/846,876 US6309601B1 (en) 1993-11-01 1997-05-01 Scanning optical detection system
US08/855,058 US6048690A (en) 1991-11-07 1997-05-14 Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis
US08/859,644 US6582660B1 (en) 1993-11-01 1997-05-20 Control system for active programmable electronic microbiology system
US08/986,065 US6051380A (en) 1993-11-01 1997-12-05 Methods and procedures for molecular biological analysis and diagnostics
US09/141,286 US6245508B1 (en) 1993-11-01 1998-08-27 Method for fingerprinting utilizing an electronically addressable array
HK98111839A HK1011079A1 (en) 1993-11-01 1998-11-09 Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics
US09/203,935 US6319472B1 (en) 1993-11-01 1998-12-02 System including functionally separated regions in electrophoretic system
US09/204,324 US6638482B1 (en) 1993-11-01 1998-12-02 Reconfigurable detection and analysis apparatus and method
US09/203,725 US6309602B1 (en) 1993-11-01 1998-12-02 Stacked, reconfigurable system for electrophoretic transport of charged materials
US09/203,730 US6375899B1 (en) 1993-11-01 1998-12-02 Electrophoretic buss for transport of charged materials in a multi-chamber system
US09/240,931 US6315953B1 (en) 1993-11-01 1999-01-29 Devices for molecular biological analysis and diagnostics including waveguides
US09/240,920 US6254827B1 (en) 1993-11-01 1999-01-29 Methods for fabricating multi-component devices for molecular biological analysis and diagnostics
US09/240,489 US6225059B1 (en) 1993-11-01 1999-01-29 Advanced active electronic devices including collection electrodes for molecular biological analysis and diagnostics
US09/239,598 US6331274B1 (en) 1993-11-01 1999-01-29 Advanced active circuits and devices for molecular biological analysis and diagnostics
US09/239,569 US6068818A (en) 1993-11-01 1999-01-29 Multicomponent devices for molecular biological analysis and diagnostics
US09/291,129 US6468742B2 (en) 1993-11-01 1999-04-12 Methods for determination of single nucleic acid polymorphisms using bioelectronic microchip
US09/354,931 US6306348B1 (en) 1993-11-01 1999-07-15 Inorganic permeation layer for micro-electric device
US09/358,788 US8389212B1 (en) 1993-11-01 1999-07-22 Method for the electronic analysis of a sample oligonucleotide sequence
US09/436,311 US6569382B1 (en) 1991-11-07 1999-11-08 Methods apparatus for the electronic, homogeneous assembly and fabrication of devices
US09/444,539 US6518022B1 (en) 1993-11-01 1999-11-22 Method for enhancing the hybridization efficiency of target nucleic acids using a self-addressable, self-assembling microelectronic device
US09/490,965 US7172864B1 (en) 1993-11-01 2000-01-24 Methods for electronically-controlled enzymatic reactions
US09/527,069 US6423271B1 (en) 1993-11-01 2000-03-16 Laminated assembly for active bioelectronic devices
US09/532,364 US6540961B1 (en) 1993-11-01 2000-03-21 Multicomponent devices for molecular biological analysis and diagnostics
US09/596,657 US6726880B1 (en) 1993-11-01 2000-06-19 Electronic device for performing active biological operations and method of using same
US09/597,866 US7101661B1 (en) 1993-11-01 2000-06-20 Apparatus for active programmable matrix devices
US09/849,119 US7425308B2 (en) 1993-11-01 2001-05-04 Systems for the active electronic control of biological reactions
US09/849,122 US7241419B2 (en) 1993-11-01 2001-05-04 Circuits for the control of output current in an electronic device for performing active biological operations
US09/912,014 US20030059929A1 (en) 1993-11-01 2001-07-24 Methods for electronic synthesis of complex structures
US09/927,820 US20010052976A1 (en) 1993-11-01 2001-08-09 Scanning optical detection system
US09/952,459 US6821729B2 (en) 1993-11-01 2001-09-12 Devices for molecular biological analysis and diagnostics including waveguides
US10/029,472 US20020085954A1 (en) 1993-11-01 2001-10-22 Inorganic permeation layer for micro-electric device
US10/170,172 US20030190632A1 (en) 1993-11-01 2002-06-11 Method for enhancing the hybridization efficiency of target nucleic acids using a self-addressable, self-assembling microelectronic device
US10/245,206 US7582421B2 (en) 1993-11-01 2002-09-16 Methods for determination of single nucleic acid polymorphisms using a bioelectronic microchip
US10/371,066 US20030162214A1 (en) 1993-11-01 2003-02-21 Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics
US10/686,440 US20040077074A1 (en) 1993-11-01 2003-10-14 Multi-chambered analysis device
US11/401,713 US8630807B2 (en) 1993-11-01 2006-04-11 Methods for the electronic, homogeneous assembly and fabrication of devices
US11/505,679 US7704726B2 (en) 1993-11-01 2006-08-17 Active programmable matrix devices
JP2006273187A JP2007057540A (ja) 1993-11-01 2006-10-04 分子生物学的分析および診断用の自己アドレス可能、自己組立て小型電子システムおよびデバイス
US11/726,520 US8114589B2 (en) 1993-11-01 2007-03-22 Self-addressable self-assembling microelectronic integrated systems, component devices, mechanisms, methods, and procedures for molecular biological analysis and diagnostics
US11/775,724 US7858034B2 (en) 1993-11-01 2007-07-10 Circuits for the control of output current in an electronic device for performing active biological operations
US11/963,317 US8313940B2 (en) 1993-11-01 2007-12-21 Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics

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US08/146,504 US5605662A (en) 1993-11-01 1993-11-01 Active programmable electronic devices for molecular biological analysis and diagnostics

Related Parent Applications (8)

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US08/677,305 Continuation US5965452A (en) 1993-11-01 1996-07-09 Multiplexed active biologic array
US08/703,601 Continuation-In-Part US5849489A (en) 1991-11-07 1996-08-23 Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system
US08/709,358 Continuation US6129828A (en) 1993-11-01 1996-09-06 Apparatus and methods for active biological sample preparation
US70826296A Continuation 1993-11-01 1996-09-06
US70826296A Continuation-In-Part 1993-11-01 1996-09-06
US08/725,976 Continuation US5929208A (en) 1993-11-01 1996-10-04 Methods for electronic synthesis of polymers
US08/846,876 Continuation US6309601B1 (en) 1993-11-01 1997-05-01 Scanning optical detection system
US08859044 Continuation-In-Part 1997-05-20

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US08/271,882 Continuation-In-Part US6017696A (en) 1991-11-07 1994-07-07 Methods for electronic stringency control for molecular biological analysis and diagnostics
US08/725,976 Continuation US5929208A (en) 1993-11-01 1996-10-04 Methods for electronic synthesis of polymers

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US08/725,976 Expired - Lifetime US5929208A (en) 1993-11-01 1996-10-04 Methods for electronic synthesis of polymers
US09/358,788 Expired - Fee Related US8389212B1 (en) 1993-11-01 1999-07-22 Method for the electronic analysis of a sample oligonucleotide sequence

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US09/358,788 Expired - Fee Related US8389212B1 (en) 1993-11-01 1999-07-22 Method for the electronic analysis of a sample oligonucleotide sequence

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AT (1) ATE211259T1 (sv)
AU (1) AU692800B2 (sv)
BR (1) BR9407952A (sv)
CA (1) CA2175483C (sv)
DE (1) DE69429535T2 (sv)
DK (1) DK0727045T3 (sv)
ES (1) ES2170133T3 (sv)
FI (1) FI961843A (sv)
HK (1) HK1011079A1 (sv)
NZ (3) NZ275962A (sv)
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EP0727045B1 (en) 2001-12-19
FI961843A (sv) 1996-06-20
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ES2170133T3 (es) 2002-08-01
EP1120469A3 (en) 2001-10-24
EP0727045A1 (en) 1996-08-21
WO1995012808A1 (en) 1995-05-11
CA2175483C (en) 2004-11-30
US5929208A (en) 1999-07-27
AU692800B2 (en) 1998-06-18
EP1120469A2 (en) 2001-08-01
NZ330037A (en) 2000-01-28
NZ275962A (en) 1998-07-28
EP1120157A3 (en) 2001-10-24
EP1120156A3 (en) 2001-10-24
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EP1120155A2 (en) 2001-08-01
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EP0727045A4 (en) 1998-04-22
FI961843A0 (sv) 1996-04-30
HK1011079A1 (en) 1999-07-02
JP2007057540A (ja) 2007-03-08
CA2175483A1 (en) 1995-05-11
BR9407952A (pt) 1996-11-26
PT727045E (pt) 2002-06-28
CN1141078A (zh) 1997-01-22
DE69429535D1 (de) 2002-01-31
DE69429535T2 (de) 2002-08-22
CN1558207A (zh) 2004-12-29
NZ330036A (en) 2000-01-28
AU8125794A (en) 1995-05-23
US8389212B1 (en) 2013-03-05
EP1120156A2 (en) 2001-08-01
DK0727045T3 (da) 2002-04-15
CN1920055A (zh) 2007-02-28

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